As their first application for veterinary health, the two PI?s have already remarkably employed their novel, patented superagonist and neoglycosylation technology to develop the first recombinant, much more potent and long-acting bovine FSH antagonists for improved cattle reproduction and a potential $30-40 Million annual market. This work was supported by a previous phase 1 SBIR from FDA-CVM which proved vital in obtaining required data for an FDA NADA and ultimately for a large licensing & commercialization agreement with Zoetis, the world?s leading veterinary health company. The current proposal of the first recombinant, much more potent and long acting equine chorionic gonadotropin (eCG) agonists for improved and more ethical reproduction in pigs and cattle is estimated both by Trophogen and potential partnering veterinary pharma companies to be an even larger annual worldwide market of $50-100 Million, a potential veterinary ?blockbuster?! In view of the PI?s remarkable success in developing and commercializing its first veterinary product with support of a phase 1 SBIR from FDA and validation of its technology and the potential market value of its reproductive products by world leader Zoetis, we hope that the current submission will receive expedited approval. The goal of this Phase 1 project is to develop and evaluate new recombinant eCG analogs as replacement for pregnant mare?s serum gonadotropin (PMSG) and P.G. 600 (PMSG+hCG). The use of PMSG obtained by inhumane bleeding of pregnant mares between days 40 and 130 of gestation is highly controversial and an inhumane business of PMSG production raises increasing concerns of the public and animals rights group. There are many limitations of current PMSG and related products including presence of horse serum contaminants in PMSG preparations, potential infection with equine viruses and prions, as well as immunological responses to highly heterogenous preparations produced in heterologous species (horse or human). There are no approved recombinant veterinary eCG products and the PIs were highly encouraged by increasing interest from major veterinary companies in their totally novel more potent and longer-acting recombinant gonadotropins, including new eCG analogs. In this Phase 1 we plan to: Aim1A Develop novel high affinity recombinant eCG superagonist analogs by minimal 4 arginine site-directed mutagenesis to produce a more potent and efficacious dual FSH + LH/CG activity molecule; Aim1B For selected eCG superagonists also engineer a novel minimal length amino acid insert containing one or two complex carbohydrate neoglycosylation sites to further increase half-life and produce analogs without reducing increased superagonist potency/efficacy; Aim 2 Optimize production and purification of these novel analogs using stable highly expressing Chinese hamster ovary (CHO) cell lines and low cost production methods in roller bottles; Aim 3 Select the best 2-4 analog candidates and characterize them in vitro by previously validated eCG analog ELISA immunoassay, SDS-PAGE electrophoresis, isoelectric focusing (IEF) gel analysis and robust in vitro FSH and LH/CG bioassays; Aim 4 Select the final eCG analog using the most important parameter determining in vivo performance: pharmacokinetic (PK) studies in mice; Aim 5 Establish CHO research cell bank (RCB) providing high expression of the lead, optimally neoglycosylated eCG analog sufficient for more expensive bioreactor production and all extensive rodent, pig and cattle studies in Phase 2.